The pivotal role of nuclear factor erythroid 2-related factor 2 in diabetes-induced endothelial dysfunction

Journal Pre-proof THE PIVOTAL ROLE OF NUCLEAR FACTOR ERYTHROID 2-RELATED FACTOR 2 IN DIABETES-INDUCED ENDOTHELIAL DYSFUNCTION Karan Naresh Amin, Elango Bhakkiyalakshmi, Jayasuriya Ravichandran, D.V.L. Sarada, Ramkumar Kunka Mohanram

PII:

S1043-6618(19)32336-9

DOI:

https://doi.org/10.1016/j.phrs.2019.104601

Reference:

YPHRS 104601

To appear in:

Pharmacological Research

Received Date:

19 October 2019

Revised Date:

23 November 2019

Accepted Date:

11 December 2019

Please cite this article as: Amin KN, Bhakkiyalakshmi E, Ravichandran J, Sarada DVL, Mohanram RK, THE PIVOTAL ROLE OF NUCLEAR FACTOR ERYTHROID 2-RELATED FACTOR 2 IN DIABETES-INDUCED ENDOTHELIAL DYSFUNCTION, Pharmacological Research (2019), doi: https://doi.org/10.1016/j.phrs.2019.104601

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THE PIVOTAL ROLE OF NUCLEAR FACTOR ERYTHROID 2-RELATED FACTOR 2 IN DIABETESINDUCED ENDOTHELIAL DYSFUNCTION

Running title: Role of Nrf2 activators in endothelial dysfunction

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Karan Naresh Amin1,2, Elango Bhakkiyalakshmi1,2, Jayasuriya Ravichandran1,2, D.V.L. Sarada2, Ramkumar Kunka Mohanram1,2* [email protected]

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Life Science Division, SRM Research Institute, SRM Institute of Science & Technology, Kattankulathur-603 203, Tamilnadu, India. 2

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Department of Biotechnology, School of Bioengineering, SRM Institute of Science & Technology, Kattankulathur-603 203, Tamil Nadu, India

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Corresponding author: Dr. K.M. Ramkumar, SRM Research Institute, SRM Institute of Science &

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Technology, Kattankulathur – 603 203, Tamilnadu

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Tel: +91-9940737854 Fax: +91-44-2745 2343

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Graphical abstract

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ABSTRACT

Endothelial dysfunction (ED) is a key event in the onset and progression of vascular complications associated with diabetes. Regulation of endothelial function and the underlying

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signaling mechanisms in the progression of diabetes-induced vascular complications have been well established. Recent studies indicate that increased oxidative stress is an important determinant of endothelial injury and patients with hypertension display ED mediated by impaired Nitric Oxide (NO) availability. Further, oxidative stress is known to be associated with inflammation and ED in vascular remodeling and diabetes-associated hypertension. Numerous

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strategies have been developed to improve the function of endothelial cells and increasing number of evidences highlight the indispensable role of antioxidants in modulation of endothelium-dependent vasodilation responses. Nuclear factor Erythroid 2-related factor 2 (Nrf2), is the principal transcriptional regulator, that is central in mediating oxidative stress signal response. Having unequivocally established the relationship between T2DM and oxidative stress, the pivotal role of Nrf2/Keap1/ARE network, has taken the center stage as 2

target for developing therapies towards maintaining the cellular redox environment. Several activators of Nrf2 are known to combat diabetes induced endothelial dysfunction and few are currently in clinical trials. Focusing on their therapeutic value in diabetes-induced endothelial dysfunction, this review highlights some natural and synthetic molecules that are involved in the modulation of the Nrf2/Keap1/ARE network and its underlying molecular mechanisms in the regulation of ED. Further emphasis is also laid on the therapeutic benefits of directly upregulating NRF2-mediated antioxidant defences in regulating endothelial redox homeostasis for

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countering diabetes induced ED. ABBREVIATIONS:

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AGEs, advanced glycation end products; AMPK, AMP-activated protein kinase; β-TrCP, Betatransducin repeats-containing proteins; CA, Cinnamic aldehyde; ECM, Extra cellular matrix; ECs, Endothelial cells; ED, Endothelial dysfunction; eNOS, endothelial nitric oxide synthase; ERK, extracellular signal-regulated protein kinase; ET-1, Endothelin-1; GCLC, Glutamate-cysteine ligase catalytic; GST, glutathione S-transferase; HDL, high-density lipoprotein; HO-1. heme oxygenase-1 ; IL-6, Interleukin 6; IL-1β, Interleukin 1β ; JNK, c-jun N-terminal kinase; Keap1, Kelch-like ECH-associated protein 1; Maf, musculoaponeurotic fibrosarcoma; NLRP3, Nod-like receptor protein 3; NO∙, nitric oxide; NQO-1, NADPH quinone oxidoreductase; Nrf2, Nuclear factor Erythroid 2-related factor 2; PI3K, Akt/Phosphatidylinositol 3 Kinases; PKC, Protein Kinase C; RES, Resveratrol; ROS, reactive oxygen species; SOD, superoxide dismutase; STZ, streptozotocin; tBHQ, Tertiery butylhydroquinone; TGF-β1, Transforming growth factor β1; TNF- α, Tumor Necrosis Factor α; VCAM, vascular cell adhesion molecule; VEGF, vascular endothelial growth factor; VSMC, vascular smooth muscle cells. KEYWORDS: Endothelial dysfunction, Diabetes, Nrf2, Keap1, Nrf2 activators

1. INTRODUCTION

Blood vessels are lined by specialized single-celled-thick layer, referred to as endothelial

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cells or endothelium. Endothelium also forms the lining in inner walls of heart chambers and lymphatic vessels. Endothelium primarily performs four functions: (i) regulates the flow of blood through the vessels, aided by its antithrombic surface, facilitating movement of plasma and cells and (ii) maintains the permeability of vessel walls, (iii) promotes angiogenesis [1], (iv) actively participates in regulation of the inflammatory processes. Endothelium functions as boundary between the body tissues and blood. Further being selectively permeable, 3

endothelium allows exchange of only certain types of chemicals and white blood cells between blood and different target tissues and/or waste materials and carbon-dioxide from tissues to blood [2] [3]. 2. ENDOTHELIAL DYSFUNCTION (ED) The healthy endothelium dictates endothelium-dependent vasodilation and suppression of hypertrophy, thrombosis and inflammation of vascular tissues [4]. Important determinant of altered vascular reactivity is the failure of endothelium in maintaining vascular homeostasis, a

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condition referred to as ‘Endothelial Dysfunction’ [2]. Early pathophysiology in vascular complications arises due to endothelial dysfunction. Endothelium released vasoactive factors

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including vasodilatory factors such as nitric oxide (NO), prostacyclin (PGI2) and endothelium derived hyperpolarizing factor (EDHF) or vasoconstrictive factors such as thromboxane (TXA2)

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and endothelin-1 (ET-1) regulate several physiological processes including cellular growth, homeostasis, inflammation and vessel wall tone [5]. NO derived from endothelium is an

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important signaling molecule and reduction in its bioactivity is considered as the hallmark of endothelial dysfunction [6]. Endothelial dysfunction is prominent in several diseases, including,

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metabolic disorders such as diabetes, hypertension and physical inactivity. 2.1 ENDOTHELIAL DYSFUNCTION AND VASCULAR COMPLICATIONS IN DIABETES Internal lumen of vasculature is lined by endothelial cells (ECs) which serve as a barrier

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between blood and smooth muscle cells that line the vasculature. ECs maintain vessel wall integrity and blood fluidity to avoid bleeding during injury [7]. In physiological as well as pathological conditions, ECs regulate both the basal tone and reactivity of the vasculature and also participate in angiogenesis. They release vaso-relaxing factors, such as NO, prostacyclin and vaso-contracting factors like endothelin-1 (ET-1) [8], which are mediators of inflammation

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and growth of vascular smooth muscle cell (VSMC). Imbalances between vasodilatation and vasoconstriction resulting in vessel wall damage lead to ED which is an early indicator of several vascular complications [5]. The loss of regulatory functions of the endothelium therefore initiate the development of vascular complications in variety of pathological conditions including diabetic micro- and macrovascular diseases. According to the DISCOVER, a global, prospective, observational study program conducted across 38 countries, the 4

prevalence of diabetes induced endothelial dysfunction was estimated as 12.7% (microvascular) and 18.8% (macrovascular) globally by 2018 [9]. The principal connect between diabetes and its associated vascular complications is blood glucose level [10]. Loss of endothelium-dependent vasodilatation resulting in slow recovery has been reported in brachial artery of diabetic patients in glucose tolerance tests [11]. Hyperglycemia with endothelial abnormalities is a determinant of chronic diabetic complications as evidenced in several clinical trials [12]. Both microvascular complications

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including retinopathy, nephropathy and neuropathy, and macrovascular complications manifested in ischemic heart disease, peripheral vascular disease and stroke develop in

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diabetes due to ED [13]. Regulation of angiogenesis, one of the essential functions of ECs

remains to be the major contributor, with excessive angiogenesis leading to retinopathy and

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nephropathy while limited angiogenesis leading to embryonic vasculopathy, impaired collateral vessel formation, delayed wound healing, neuropathy and transplant rejection [14].

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Regulation of endothelial function and the underlying signaling mechanisms in the progression of diabetes-induced vascular complications have been understood. Hyperglycemia

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and generation of excessive ROS cause aggravation of cellular injury by activating several metabolic pathways including auto oxidation of glucose resulting in the formation of ketoaldehydes, glycotoxins or advanced glycation end products (AGEs) and polyols and

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stimulation of the eicosanoid metabolism [15] [16]. Endothelial markers including, ET-1, selectins, thrombomodulin, type IV collagen and Von Willebrand factor are reported to increase in hyperglycemic states resulting in poor vasodilatation [15] [17]. In addition, the expression of AGEs, angiotensin II (ANG-II), plasminogen activity inhibitor-1 (PAI-1), and protein kinase C (PKC), the key cell signaling modulators are also perturbed during diabetic conditions

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[18]. These molecular changes culminate in inducing structural and functional changes in the vasculature leading to the advancement of the disease. 3. ENDOTHELIAL DYSFUNCTION-RELATED PATHOPHYSIOLOGICAL ABNORMALITIES IN DIABETIC COMPLICATIONS 3.1 Diabetic Cardiomyopathy

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Diabetic cardiomyopathy, a microvascular disease that is the cause for cardiovascular complications, mainly results due to vascular endothelial cell dysfunction [19] and is one of the major cause of death in diabetic population [20]. Pathologically, it is characterized by the development of hypertrophy of cardiomyocyte, fibrosis and cardiomyocyte apoptosis and early impairment of diastolic function. Impaired blood flow to the heart muscle, causes elaboration of endothelial cell matrix proteins resulting in basement membrane thickening and interstitial fibrosis [21] [22].

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The fact that ET-1 causes vasoconstriction in target organs has been clearly established. Such altered vasoregulation is correlated with reduced availability of NO resulting from

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oxidative stress-mediated sequestration of NO. Reduction in NO and increase in ET-1 transduce signals to the contractile cells and vascular endothelial cells respectively. This results in myocyte

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hypertrophy and increased expression of proteins of endothelial cell matrix [22] [20] culminating in basement membrane thickening, these conditions along with reduced flow of

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blood, produce ischemia in the cardiac tissue resulting in focal scarring [23]. Vasoactive factors such as ET-1 and NO should therefore be at the forefront while developing therapies to reduce

3.2 Diabetic Nephropathy

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the vascular complications in hyperglycemia.

Maintenance of constant volume of plasma and extracellular fluids, salt concentration

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and pH balance are the primary functions of kidney. Membranes of the glomerular capsule filter out plasma proteins including albumin, with less than 1% escaping the filtration process and excreted in the urine [24]. Inflammation, fibrosis and loss of renal function generally result due to increased amount of albumin in the glomerular filtrate and excessive reabsorption [25]. Early pathogenic events that cause damage to the filtration process of the kidneys include ED

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resulting from defects endothelial progenitor cells. Impairment of microcirculatory functions leading to nephropathy is the major cause of persistent kidney complications in diabetes [13]. Nephropathy involving manifestations such as microalbuminuria and hyperfiltration at

early stages followed by deterioration to end-stage renal disease is a serious microvascular complication of both type 1 and type 2 diabetes mellitus [26]. Microalbuminuria is usually the initial sign of renal complications and may progress to overt albuminuria. High intra-glomerular 6

pressure and glomerular basement membrane permeability results in albuminuria, and is indirectly influenced by interactions of ECs with mesangial cells and podocytes in a paracrine fashion [27]. Renal hyperfiltration mainly occurs due to the dilation of afferent glomerular arteriole and it is generally associated with diabetes associated vascular dysfunction leading to development of glomerulosclerosis and tubulointerstitial fibrosis [28] and decline of renal function. In non-insulin-dependent diabetes mellitus, Neri et al., identified early endothelial alterations and concluded that endothelial dysfunction occurs much earlier in patients with

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microalbuminuria [29]. Abnormal renal function also results due to reduction of antiatherogenic function of ECs resulting in ED.

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Since minor alterations in the vascular tone of the glomerular afferent arterioles

adversely influences blood flow and intensifies diabetic nephropathy, preserving the functional

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integrity of the renal system by way of monitoring the levels of vaso-modulatory molecules including vasodilators and vasoconstrictors is important. Preclinical and clinical trials in diabetic

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patients revealed the involvement of signaling molecules including, endothelial nitric oxide (eNOS/NO), ET-1, renin-angiotensin, and vascular endothelial growth factor (VEGF) [30] and

nephropathy. 3.3 Diabetic Retinopathy

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therefore need to be considered as most probable targets for drug therapies against diabetic

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Stimulation and development of microvessels in the retina due to release of angiogenic factors and ischemic conditions is known as proliferative retinopathy [16]. Alteration in the expression of cell signaling modulators including adhesion molecules, growth factors, inflammatory cytokines, neurotrophic factors and vasoactive agents, in diabetic condition results in this multifactorial disease of the retina [31]. Despite of the advancements in the

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diagnosis and therapies for complications of the eye, diabetic retinopathy is reported to be leading cause of blindness among the western working-aged populations and fifth cause across the globe [32]. It is categorized broadly into three stages: non-proliferative, pre-proliferative, and proliferative. On clinical examination, although the retina of patients with prolonged diabetes might appear normal, several significant histological and biochemical changes such as, basement membrane thickening, adhesion of leukocytes, pericyte loss, vascular alterations, 7

blood–retinal barrier damage and neovascularization are known to occur [33]. Alterations in the thickening of basement membranes of capillaries and loss of perivascular cells results from progressive dysfunction of ECs resulting in changes in retinal morphology and physiology [34].

3.4 Diabetic Neuropathy Development of neuropathy results when the supply of blood through vasa nervorum is not adequate. Imbalances in neuron metabolism and impaired nerve blood flow, both

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culminate in diabetic neuropathy. Owing to its complexity, the correlation between ED and diabetic neuropathy is yet to be delineated. The course of diabetic neuropathy is reported to

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be accelerated by dysfunction of EPCs, which normally maintain the homeostasis of nutritive micro-vasculature [35]. Insights into the underlying similarities in the cellular anomalies

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occurring both in endothelial cells and neurons indicate that mechanisms involved overlap with each other in diabetic neuropathy and diabetes-associated microangiopathy, and hence the

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former is often used as index of the later [36, 37] and is proven that ED is sufficient to cause neuropathy [38]. Cardiovascular risk and diabetic neuropathy in diabetes has been reported to

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be mediated by ED according to a study conducted by Roustit et al [39]. Diabetes and Endothelial Dysfunction: A Clinical Perspective Many pharmacological therapies with different targets are used for the treatment of ED.

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Usually cardiovascular agents like calcium agonists, beta blockers, statins, erythropoietin, angiotensin converting enzyme (ACE) inhibitors, angiotensin receptor blockers (ARBs) and renin inhibitors are being used to improve endothelial functions [40]. Among various therapies, ACE inhibitors and ARBs have proven to be a promising therapy in the treatment of ED. Hermann et al., reported that the ACE inhibitor, Quinapril increases insulin-stimulated endothelial function

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and adiponectin gene expression with concomitant the reduction in systolic and diastolic blood pressure in patients with type 2 diabetes [41]. Few drugs including Captopril, Enalapril, Perindopril and Lisinopril resulted in increased endothelium dependent vasodilation [42] [43] [44] [45]. ARBs such as Irbesartan, Telmisartan, Valsartan and Olmesartan have been shown to improve endothelial function. [46] [47] [48] [49]. In addition, glitazone family of drugs such as Pioglitazone, Troglitazone and Rotiglitazone also improved the function of endothelium in 8

diabetic patients [50] [51]. Endothelial nitric oxide synthase (eNOS) enhancers are one of the novel therapeutics for the treatment of ED, which induce and upregulate eNOS transcription. Also, there are reports of endothelial progenitor cells (EPCs) resulting in improved vessel repair, vascularization and tissue perfusion in patients with peripheral vascular diseases [52]. 4. OXIDATIVE STRESS AND ENDOTHELIAL DYSFUNCTION Free radicals are important signaling molecules in the regulation of vascular homeostasis and the primary stimulus of vascular dysfunction [53]. Imbalance between

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production of free radicals and the ability of antioxidant molecules to quench them results in oxidative stress as the free radicals outnumber the quenching capacity of antioxidants [54].

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NO availability is reduced due to increased free radical production which decreases

endothelium-dependent relaxation [55]. Harmful intermediates generated by excess free

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radicals, cause alterations in structures of membranes, proteins and DNA resulting in altered metabolic activity and cellular dysfunction leading to cell death. Pathogenesis of diabetes and

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its associated complications, have often been correlated to perturbed redox homeostasis and increased free radical-induced DNA damage [56]. Other factors that contribute to ED include

conditions [57].

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endoplasmic reticulum stress (ER stress) resulting from hyperglycemic, hypoxic and shear stress

Few clinical studies reported that increased oxidative stress to be an important

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mechanism for impaired endothelial function in patients with atherosclerosis or cardiovascular risk factors. The increased superoxide production in association with endothelial vasomotor dysfunction have been reported in patients with coronary artery disease [58] [59]. Furthermore, this could be reversed by the administration of antioxidants such as vitamin C [60] [61]. On the other hand, a meta‐analysis of 68 randomized trails revealed that chronic

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treatment with antioxidants such as vitamin A, vitamin C, vitamin E, β‐carotenes, or selenium has no significant beneficial effect on vascular mortality in diabetes patients [62]. Heitzer et al reported that endothelial dysfunction and increased vascular oxidative

stress predict the risk of cardiovascular events in patients with coronary artery disease [63]. Costantino et al investigated the role of pro-apoptotic protein p66Shc in persistent ROS generation and endothelial dysfunction in patients with T2DM patients [64]. 9

Diabetic patients’ exhibit altered expression profiles of miRNAs involved in angiogenesis, vascular repair, and endothelial homeostasis. Thus, patients with T2DM exhibit low levels of circulating miRNA‐126 compared with healthy subjects. The miRNA‐146 family participates in the regulation of oxidative stress and the production of proinflammatory factors [65] [66]. Circulating levels of miR‐146a was reported to be decreased in diabetic patients [67]. 4.1. NRF2/Keap1/ARE Network. The regulation of redox homeostasis at cellular level, in both normal and diseased

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conditions occurs primarily at the transcriptional level, mediated by Nrf2/Keap1/ARE signaling pathway resulting in upregulation of antioxidant response [56]. This pathway involves more

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than hundred genes with anti-oxidant functions, improving adaptability of cells to oxidative stress environment and increasing survival. These functions include, enzymatic antioxidants,

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molecules that quench free radicals, glutathione homeostasis, reduction of inflammation, expression of phase I and II electrophile detoxifying proteins and enzymes, proteins that

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conjugate and transport of toxic substances, proteasome function, recognition of DNA damage, transcription factors, growth factors and vascular functions [68].

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Nrf2 (Nuclear factor Erythroid 2-related factor 2) is the principal regulator that mediates the oxidative stress response [69]. It binds upstream of cis-regulatory element involved in antioxidant response (ARE), and induces the transcription of several functions mentioned

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above. Under normal physiological conditions, Kelch-like ECH-associated protein 1 (Keap1) binds Nrf2 through interactions with Nrf2-ECH homologous domain (Neh2) phosphorylation site. Keap1 belongs to Cullin3- (Cul3-) based E3-ligase complex, which catalyzes the ubiquitination of Nrf2 subjecting it to proteosomal degradation [70]. Oxidative and/or electrophilic stress environment, is sensed at the cellular level by

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modification of cysteine residues in Keap1, this modification results in change in stoichiometry of thiol-reactive groups culminating not only in the dissociation of Nrf2 from Keap1, but also in stabilization of Nrf2 [71]. Stabilized Nrf2 forms heterodimers with small musculoaponeurotic fibrosarcoma (Maf) family transcription factors which translocate to the nucleus where they bind upstream of ARE, recruit other factors required for activation of a variety of antioxidant genes, including, glutathione S-transferase (GST), heme oxygenase-1 (HO-1), NADPH quinone 10

oxidoreductase (NQO-1) and superoxide dismutase (SOD1). These antioxidant enzymes quench free radicals and convert into less toxic forms by employing any one of the four primary mechanisms: (i) redox reactions, exposing the functional groups on the surface of hydrophobic molecules, (ii) trapping of nucleophiles, (iii) efflux or compartmentalization of toxic metabolites through transporter proteins, and (iv) maintenance of non-oxidant environment primarily by thiol group containing molecules [72]. This defensive regulatory mechanism of Keap1 ensures continued binding of Nrf2 during unstressed conditions or releasing and stabilizing it, in an

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oxidant environment, for induction of an appropriate antioxidant response [73] [74]. The mode of activation of Nrf2 by small molecules have been identified in which indirect interaction

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mediated mechanism through upstream kinases such as Akt/Phosphatidylinositol 3 Kinases

(PI3K), Protein Kinase C (PKC), extracellular signal-regulated protein kinase (ERK) and c-jun N-

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terminal kinase (JNK) and direct interaction mediated mechanism including cysteine modification in Keap1, Cysteine modification in Nrf2 as well as inhibition of proteosomal

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degradation of Nrf2 [75]. From our laboratory we have demonstrated a novel mode of activating Nrf2 using pterostilbene through interactions with the arginine triad residues of

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Kelch domain of Keap1 [76].

In a study on the association of the redox regulator, Nrf2 and inflammatory cytokines in patients with recent onset type 2 diabetes, the plasma Nrf2 levels were significantly

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reduced and correlated positively with Th2 cytokines, and negatively with Th1 cytokines indicating lower levels of Nrf2 to favor Th1 response [77]. The underlying mechanisms of low expression of Nrf2 in diabetes remain largely unknown. In recent years, Keap1-Nrf2 signaling network has emerged as a favorable opportunity of research, to combat diabetes and its related complications. Our laboratory, has developed a luciferase reporter complementation

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assay for screening Nrf2 activators in vitro and in vivo and identified the potential activators of Nrf2 [78]. The therapeutic prospects of Nrf2 activation in diabetes are being worked, at molecular level in both in vitro and in vivo models using activators of Nrf2 including, Morin, Naringenin, Pterostilbene, Quercetin and Resveratrol [79-82]. Protection of pancreatic β-cells from oxidative damage and cytokine stress, in addition to normalization of altered glucose metabolism in diabetic animals are being addressed. 11

4.2 ROLE OF NRF2 IN DIABETES-ASSOCIATED ENDOTHELIAL DYSFUNCTION Having unequivocally established the relationship between T2DM and oxidative stress, the pivotal role of Nrf2/Keap1/ARE network has become a target for developing therapies towards reducing the oxidant environment in the cells. Accumulated evidences suggested that

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a perturbation to this antioxidant network is often coupled with the pathophysiology of diabetes and its protracted complications [83].

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Vascular dysfunction is central to development of vascular complications associated with diabetes, the major contributors being increased inflammation and oxidative stress [84].

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Due to excessive generation of both O2− and H2O2 in the endothelium of diabetic subjects, intracellular production of hydroxyl radicals is enhanced. Increases in vascular endothelial

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permeability due to oxidative stress promotes leukocyte adhesion, coupled with alterations in redox-regulated transcription factors and endothelial signal transduction [16]. In diabetic

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subjects, vasodilation is often associated with the impaired action of nitric oxide [85] [86]. In pathogenic conditions, there is a direct implication of involvement of oxidative processes in the induction of inflammatory molecules in vascular remodeling and inflammation

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causing changes in the vasculature [87]. An important pro-atherosclerotic cell adhesion molecule vascular cell adhesion molecule 1 (VCAM-1) is reported to be regulated by the Nrf2 signaling pathway [88]. Most of the recent research evidences hinge towards targeting Nrf2 signaling network pathway in averting diabetes-mediated endothelial dysfunction. Inflammation plays a crucial role in the etiology of vascular disease in diabetes.

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Hyperglycemia induces inflammation through various mechanisms. The increased generation of free radicals in endothelial cells during hyperglycemic condition leads to the activation of major signaling cascades involved in the pathogenesis of diabetic complications such as polyol pathway, formation of AGEs, expression of the receptor for AGEs, activation of PKC isoforms and increased hexosamine pathway [43]. It has also been reported that hyperglycemia increases toll-like receptor (TLR) activity through an increased free radical production 12

augmenting inflammation. Moreover, the increased oxidative stress has been reported to diminish the bioavailability of nitric oxide, resulting in vascular impairment. In addition, it has been reported that redox-dependent activation of endothelial Nod-like receptor protein 3 (NLRP3) inflammasomes in endothelial cells is an important cause hyperglycemia-induced endothelial dysfunction [61]. In diabetes, lipids aggravate the inflammatory process by promoting oxidative stress and leukocyte activation and ultimately foster endothelial dysfunction and atherosclerosis progression.

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Hyperglycemia increases the activity of transcription factor, nuclear factor-kappaB (NFkB), in endothelial cells and promotes the activation of pro-inflammatory cytokines,

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chemokines, the expression of adhesion molecules and activation of inducible nitric oxide synthase (iNOS). It has been evidenced that inflammatory cytokines increase vascular

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permeability, alter vasoregulatory responses, increase leukocyte adhesion to endothelium, and facilitate thrombus formation. Few studies evidenced the anti-inflammatory properties of Nrf2

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driven through its ability to negatively regulate NF-kB.

5. Nrf2 ACTIVATORS IN AMELIORATING DIABETES INDUCED ED

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Vascular complications in the pathogenesis of diabetes result from interactions between autophagy, inflammation, metabolism, and oxidative stress. Cytoprotective contributions of Nrf2 as a stress sensitive and ameliorative molecule extend beyond the antioxidant defense

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[89] and therefore Nrf2 activators remains to be promising in restoring endothelial function in diabetes. Hitherto, several activators of Nrf2 are known to combat diabetes induced ED (Figure 1), very few are currently in clinical trials. Focusing on their therapeutic value in diabetesinduced endothelial dysfunction, the following sections will highlight some natural and synthetic molecules that are involved in the modulation of the Nrf2/Keap1/ARE network and

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their mode of action (table 2).

5.1 Sulforaphane

1-isothiocyanato-(4R) methyl sulfinyl butane, commonly referred to as isothiocyanate

sulforaphane (SFN) is an organosulfur compound rich in several cruciferous vegetables including broccoli, brussels sprouts or cabbages. Several studies highlight the protection

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conferred by this compound in diabetes and in cardiovascular and neurodegenerative diseases [90]. Nrf2 signaling has been linked to the effect of SFN in diabetics as highlighted in several studies. ARE-linked gene expression was induced by SFN mediated nuclear translocation of Nrf2 [91]. Accumulation of methylglyoxal and its excretion was reduced by SFN, further activation of the hexosamine and PKC pathways was observed in diabetic subjects. [91]. SFN is proved to prevent biochemical dysfunction and related functional responses of endothelial cells during

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hyperglycemia. Pereira et al., reported improved endothelium-dependent vascular relaxation in aorta of diabetic rats administered with SFN. Consistent with its documented effects in other

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tissues, SFN in both aorta and mesenteric arteries of diabetic rats, produced a significant

increase in Nrf2 expression, suggesting that SNF promotes both increased expression and

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nuclear translocation [92].

SFN improved metabolic profile and reduced renal injury in mice with streptozotocin-

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induced diabetes SNF is reported to reduce ROS and attenuate induction of the extra cellular matrix (ECM) proteins, growth inhibitory protein p21 and profibrotic mediator transforming

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growth factor-β in kidney tissues [93]. A study by Cui et al., showed elevated renal Nrf2 expression and prevented diabetic nephropathy, reduced renal inflammation, oxidative damage and fibrosis in diabetic group receiving SFN treatment over a period of three months [94].

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Recently high glucose-induced retinal inflammation and oxidative stress are reported to be attenuated by SFN, further it is also known to activate the Nrf2 network and inhibit the NLRP3 inflammasome formation both in vitro and in vivo [95]. SFN supplementation resulted in nuclear translocation and accumulation of Nrf2, and enhanced expression of HO1 and NQO1 in injured retina of diabetic rats [95]. Wang et al., explored the effect of SFN on T2DM-induced

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pathogenic changes in aorta and reported increase in inflammation, oxidative/nitrative stress, thickening and structural derangement of the wall, fibrosis and apoptosis in aorta of diabetic mice [96], however, SFN treatment resulted in significant upregulation of Nrf2 expression and attenuated these changes. 5.2 Curcumin

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Curcumin (CUR) is a popular spice, turmeric used in Asian cuisine and is beneficial against diabetes and its related complications have been reported. The principal curcuminoid found in turmeric has anti-oxidant and anti-inflammatory properties [128]. He et al., reported the suppression of NF-κB signaling pathway by CUR, resulting in defense against inflammation, hypertrophy and fibrosis in the heart. Levels of glucose, glycosylated hemoglobin, inflammatory cytokines such as Interleukin 6 (IL-6), Tumor Necrosis Factor α (TNF-α) and monocyte chemoattractant protein-1 (MCP-1) were reduced in diabetic

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animals fed with CUR supplemented diet [129]. Similarly, accumulation of AGEs, Interleukin 1β (IL-1β) and TNF-α decreased, diabetes-induced interstitial fibrosis, cardiomyocyte hypertrophy

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and left ventricular dysfunction, were all reversed by administration of CUR to diabetic rats [104].

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CUR was observed to prevent a series of events, namely, downregulation of Ecadherin and upregulation of α-smooth muscle actin, associated with the Nrf2-activation and

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subsequent HO1 induction during epithelial-mesenchymal transition in the rat kidney tubular epithelial cell line [130]. Additionally, the inhibition of transforming growth factor β1

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(TGF-β1), fibronectin, and collagen IV were also involved in the mechanism by which CUR protects the vasculature against diabetic nephropathy. Few CUR derivatives have also proved to be effective in ameliorating diabetic

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nephropathy. Wang et al., reported that administration of CUR analogue, C66 resulted in reduction of glomerulosclerosis and tubulointerstitial fibrosis mediated by Nrf2 signaling in diabetic mice. Further, C66, inhibited the JNK signaling pathway, reduced cardiac inflammation and also improved cardiac function, in addition to reducing serum and heart hypertriglyceridemia. C66 also caused inactivation of NF-κB and inhibited pro-inflammatory

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cytokines induced by high glucose [131]. In streptozotocin-induced diabetic mouse model, J17 a molecule with structural similarities to CUR, resulted in reduction in inflammation and fibrosis in H9C2 cardiomyocytes [105], the effect was attributed to perturbation of the P38 and AKT signaling pathways. 5.3 Bardoxolone methyl (CDDO-Me/RTA 402)

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Bardoxolone methyl, CDDO, and CDDO-imidazolide are synthetic triterpenoids, and powerful inducers of Nrf2/ARE signaling network which have been used against several diseases related to oxidative damage and inflammation [96]. In a recent clinical trial, end stage renal disease patients with T2DM were grouped and administered bardoxolone methyl to determine whether it would reduce the risk of stage 4 chronic kidney diseases. Patients administered with bardoxolone methyl group, showed better kidney function and reduced concentrations of serum creatinine, urea nitrogen and uric acid

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[108], but albuminuria increased. The degree of albuminuria is an indicator of glomerular membrane integrity and vascular homeostasis used as an index of kidney function and in

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assessment of risk of cardiovascular complications as CKD progresses. The trial was terminated due to safety concerns, as increase in cardiovascular events were reported in the bardoxolone

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methyl group [110].

Dihydro-CDDO-trifluoroethyl amide (dh404), a synthetic triterpenoid derivative

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saturated the binding capacity of Keap1 and interrupted the integrity of Keap1-Cul3-Rbx1 E3 ligase complex-mediated ubiquitination of Nrf2 and its subsequent degradation, thereby

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rendering more Nrf2 to be translocation into the nuclei and activation of Nrf2-driven gene transcription. Dh404 prevented diabetes-induced oxidative damage in cardiac tissues and insulin resistance in streptozotocin (STZ)-induced diabetic mice by activation of cardiac Nrf2

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[111]. Sharma et al., demonstrated that Nrf2 activation via dh404 improves endothelial function by targeting pro-oxidative and pro-inflammatory pathways in the Akita mouse, a model of hyperglycemia and hypoinsulinemia [109]. 5.4 Tertiery butylhydroquinone

Tertiery butylhydroquinone (tBHQ) is used as a preservative due to its highly strong

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antioxidant properties [132]. Auto-oxidation of tBHQ generates tert-butyl benzoquinone (TBQ), an electrophilic metabolite. TBQ is known to bind covalently to cysteine residues in Keap1 and hence releasing Nrf2 [133]. It is reported that tBHQ increases the expression of Nrf2 and its downstream target antioxidants genes, including γ Glutamyl Cysteine Synthase (GCS) and HO-1 resulting in reduction of hyperglycemia-induced kidney injury. [112]. Glucose-induced anomalies in Nrf2- Glutamate-cysteine ligase catalytic (GCLC) subunit were regularized by tBHQ 16

in diabetic retinopathy impinging its utility as therapeutic compound against diabetic retinopathy [134]. In STZ diabetic rats, tBHQ treatment resulted in increased Nrf2 activity in macrophages and vascular smooth muscle cells within atherosclerotic lesions. Further reduction both in size of atheroma plaques and amount of lipid and attenuation of inflammation by reducing macrophage load in the lesions, foam cell size and chemokine expression were observed [89] indicating its protective role diabetic cardiomyopathy. Administration of tBHQ in diabetic mice

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revealed enhanced Nrf2 activity in macrophages and VSMCs within atherosclerotic lesions indicating a key role in attenuating atherogenesis.

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Feeding STZ-induced diabetic mice with standard pellet diet containing 1% tBHQ, not only decreased extracellular matrix deposition and malondialdehyde compared to the diabetic

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mice fed with regular diet but also caused activation of Nrf2 and expression of its downstream genes in the glomeruli. Glomerular morphology and function and eNOS expression in the renal

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microvessel endothelium were preserved in the kidney tissues of diabetic animals [113]. These studies indicate that tBHQ administration associated Nrf2 activation can provide a coordinated

5.5 Cinnamic aldehyde

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regulation to protect endothelial function in diabetic nephropathy.

90% of the essential oil isolated from bark of cinnamon is Cinnamic aldehyde (CA) [135].

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It is a straw coloured viscous liquid that gives flavor and odour. CA possess anti-cancer antiinflammatory, anti-microbial, anti-oxidant and anti-ulcer properties along with hypoglycemic and hypolipidemic potentials [136]. Reduction in the levels of glucose, triglycerides and total cholesterol and elevation in the levels of high-density lipoprotein (HDL)-cholesterol are reported both in STZ-induced diabetic rats and db/db transgenic mice administered orally with

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of CA [137]. Hypoglycemic and hypolipidemic effects have been proved by structure–activity relationship studies, α-β-unsaturated aldehyde functional group in CA binds covalently with thiol groups of Keap1 releasing and stabilizing Nrf2 [138]. Zheng et al., provided experimental evidence for Nrf2-mediated protection through the negative regulation of TGF-β1 and p21 and reported that CA ameliorated albuminuria and minimized renal damage induced by hyperglycaemia in an Nrf2-dependent fashion [93] and 17

suggested the use of CA in the management of metabolic disorders and improving renal function. Wang et al., reported that in hyperglycemia, CA restores endothelial function, an effect that is mediated by Nrf2 activation and enhanced expression of its downstream targets [115]. CA administration therefore represents a promising intervention to diabetic patients with risk of vascular complications. 5.6 Resveratrol Resveratrol (RES) is a polyphenol, found in grapes and red wine [139] with antioxidant

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properties. It has been proven to possess a wide spectrum of pharmacological properties such as anti-inflammatory, anti-oxidative, anti-carcinogenic, anti-aging, neuroprotective and

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cardioprotective effects [140]. RES, is known for preventing cancer, regulating platelet

functions, and protecting from cardiovascular diseases [141], improvement of renal functions,

mouse models of age-related renal injury [142].

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for curing proteinuria, glomerulosclerosis, tubular interstitial fibrosis and inflammation in

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Administration of RES to streptozotocin–nicotinamide-induced diabetic rats attenuated hyperglycemia-mediated oxidative stress, thereby restoring the ultrastructural morphology

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such as tubulonecrosis, glomerulosclerosis, glomerular basement membrane thickening and changes in the proximal and convoluted tubules [143]. Further, RES also regulated the expression of Nrf2, and its downstream targets including GCS and HO-1 in diabetic kidney.

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Promotion of vascular health in metabolic diseases by restoration of endothelial function by RES, via TNFα-induced activation of NAD(P)H oxidase and eNOS phosphorylation, suggests its potential for developing new treatment strategies. [144]. The protective effect of RES investigated on vascular endothelium of diabetic rats identified its influence on retinal Nrf2 and HO-1 genes, that are associated with quenching of ROS [144].

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5.7 MG132

A peptide-aldehyde proteasome inhibitor MG132 (Z-Leu-Leu-Leu-CHO) is widely used in

protein degradation studies, to understand the role of proteolytic fragments in disease progression and biology for developing new therapeutic strategies [145] [146]. Luo et al., explored the beneficial effect of MG132 on diabetic nephropathy through Nrf2 activation [121]. Treatment of STZ diabetic rats with the peptide (10 μg/kg) for twelve 18

weeks resulted in (i) decrease in protein excretion in urine and reversal of renal pathological changes (ii) reduced renal 26S proteasome activity (iii) decrease in nitrosative damage to the kidneys (iv) upregulation of renal expression of Nrf2, catalase, glutathione peroxidase (GPx) and SOD1 were restored (v) while levels of NF-κB were reduced in renal tissues [121]. These findings suggest that MG132 can delay the development and progression of nephropathy in diabetic rats by inhibiting the proteasome resulting in increasing the stability of Nrf2 and there by inducing the expression of Nrf2 dependent antioxidant genes.

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26S proteasome activity is enhanced in hyperglycemic condition. MG132 administration abolished NF-κB-mediated renal and aortic inflammatory response resulting from

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peroxynitrite/superoxide-mediated PA700-dependent proteasomal activation in early diabetes [147]. MG132 also attenuated oxidative stress-induced damage by suppressing NF-κB in

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coronary arterioles thereby reducing the burden of cardiac damage in T2D mice model [148]. Several in vitro and in vivo studies have highlighted that MG132 inhibits proteasomal activity,

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thus stabilizing tissue Nrf2 levels resulting in IκB upregulation and renal protection thereby preventing diabetic nephropathy [123] [149] [146].

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5.8 Pterostilbene

Pterostilbene chemically is trans-3,5-dimethoxy-4-hydroxystilbene and finds application as antacid, anti-diarrheal and cardiotonic, [150]. It is abundant in grapes and blueberries and in

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the heartwood of sandal wood trees and leaves of grapes [151, 152]. PTS, has a close structural similarity with RES [152]. Several studies have demonstrated the preventive and therapeutic benefits against diabetes, neurodegeneration and vascular complications owing to its antioxidant activity [79, 153] [154].

Studies from our laboratory reported PTS to be an activator of Nrf2. Protection of

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pancreatic β-cells from apoptosis was rendered by PTS, in addition to improving the expression of Nrf2 downstream target genes [79]. Our studies also highlighted the reversal of the abundance of key glucose metabolism enzymes, and improved insulin secretion in diabetic mice resulting in maintenance of blood glucose levels [124]. Studies from our lab also provided convincing evidence for anti-hyperlipidemic and anti-peroxidative role of PTS via Nrf2 signaling in diabetic mice model [155]. 19

Kosuru et al., reported not only decreased cardiac hypertrophy, hypertension, enhanced myocardial oxidative stress, inflammation and NF-κB expression in fructose-fed rats, administered with PTS but also increased the expressions of AMP-activated protein kinase (AMPK, Nrf2, HO-1 in cardiac tissues, resulting in prevention of cardiac oxidative stress and inflammation [156]. High risk for cardiovascular diseases is often associated uremia-related endothelial dysfunction in chronic kidney disease. Uremic toxins including indoxyl sulfate in subjects with end-stage renal disease provokes endothelial damage. PTS is reported to be

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effective in reducing US-evoked endothelial cell dysfunction partly via the Nrf2 signaling pathway leading to reduction of oxidative/nitrative stress and inflammatory response [157].

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Conclusion

Nrf2 has been implicated in a range of toxicities and chronic diseases that are associated

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with oxidative stress and has emerged as a major regulator of oxidant resistance. Although there are reports, that dietary supplementation of natural antioxidants can effectively reduce

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oxidative stress in diabetic conditions, there is a dearth of evidence in the clinical setup, related to specific targets of these molecules or specific mechanisms activated by these molecules that

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aid in restoration of normal functioning of the endothelium. The cytoprotective capacity of Nrf2 against toxicity and chronic diseases by means of activating the antioxidant signaling network has opened new avenues for drug development. On the other hand, the distinct

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pathways that cause vascular complications in diabetes are yet to be unraveled. Therefore, a better insight into functioning of the endothelium in a diabetic environment and dissecting out the Nrf2 mediated signaling networks that operate in maintenance of vascular homeostasis would aid in appropriate utilization of the known leads in development of therapies for

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prevention and treatment of vascular diseases in diabetes.

Declarations of interest: none

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Acknowledgement The present study was supported by grants from the Science & Engineering Research Board

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(SERB) (grant no. EMR/20l6/006196).

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Figure Legends

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Figure 1. Mechanism of Nrf2 activators against hyperglycemia-induced Endothelial Dysfunction. Under basal conditions, Nrf2 is sequestered in the cytosol by Keap1 which leads to ubiquitination followed by proteasome degradation and this signaling is known as canonical pathway. On the other hand, the phosphorylation of Nrf2 by GSK-3 aids its recognition by β-TrCP, resulted in Cul1-mediated ubiquitination, followed by Nrf2 proteasome degradation and this signaling is known as non-canonical pathway. During oxidative stress, Nrf2 dissociates from Keap1 and translocates to the nucleus, where it dimerizes with members of the small musculoaponeurotic fibrosarcoma (Maf) family and binds to ARE genes such as HO-1. NQO1, GCL, GSTs, CAT, SOD, and thioredoxin UDP-glucuronosyltransferase. The mode of activation of Nrf2 by small molecules have been identified as indirect interaction mediated mechanism through upstream kinases such as Akt/Phosphatidylinositol 3 Kinases (PI3K), Protein Kinase C (PKC), extracellular signal-regulated protein kinase (ERK) and c-jun N-terminal kinase (JNK) and direct interaction mediated mechanism including cysteine modification in Keap1, Cysteine modification in Nrf2 as well as inhibition of proteosomal degradation of Nrf2.

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Table 1: Role of endothelial dysfunction in the pathophysiology of diabetic complications

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DIABETIC PATHOGENESIS COMPLICATIONS Diabetic Early impairments in diastolic function, development of Cardiomyopathy cardiomyocyte hypertrophy, myocardial fibrosis, cardiomyocyte apoptosis Diabetic High intra-glomerular pressure, glomerular basement membrane Nephropathy (GBM) permeability, albuminuria, glomerulosclerosis, tubulointerstitial fibrosis Diabetic Retinal ischemia, perivascular cell loss, blood–retinal barrier damage Retinopathy Diabetic Imbalances in neuron metabolism and impaired nerve blood flow Neuropathy

33

Table 2: Sources of Nrf2 activators and their mechanism against endothelial dysfunction

Broccoli, brussels sprouts or cabbages

Binds to the cystine specific residues of keap1 and keeps Nrf2 free for translocation [97] [98] Phosphorylation of ERK [99]

Curcumin

Turmeric

Modifies the cystine residues of keap1 [101] Hypomethylates DNA to restore Nrf2 expression [102]

Diabetic cardiomyopathy: endothelium- dependent vascular relaxation in aorta Diabetic nephropathy: reduce renal injury and inflammation

ur na

Bardoxolone methyl and its

[100] [94] [95]

Diabetic retinopathy: attenuates retinal inflammation

Diabetic cardiomyopathy: Reduces cardiac [104] hypertrophy, ventricular dysfunction, cardiomyocyte hypertrophy and interstitial fibrosis [105] Diabetic nephropathy: reduces glomerulosclerosis and tubulointerstitial fibrosis [106] Diabetic retinopathy: Ameliorates thinning of the retina, apoptosis of the retinal ganglion cells and inner nuclear layer cells, thickening of retinal capillary basement membrane and disturbance of photoreceptor cell membranous disks

lP

Phosphorylation of p38 [103]

Reference

of

Sulforaphane

Experimental Model

ro

Mode of Action

-p

Source

re

Molecule

[107]

Diabetic neuropathy: Attenuates mechanical allodynia

Synthetic triterpenoid

Modifies the cystine residues of keap1 [108] [109]

Diabetic nephropathy: Improved glomerular [110] integrity, kidney function and endothelial dysfunction [111]

Tertbutylhydroquinon e

Synthetic

Enhance the expression of Nrf2 by accumulating Nrf2 inside nucleus [112]

Diabetic cardiomyopathy: inflammatory milieu of atherosclerotic plaques

Cinnamic aldehyde

Cinnamon bark

Phosphorylation of ERK [114]

Diabetic nephropathy: ameliorate albuminuria and minimize renal damage

Jo

derivative dh404

[113]

Diabetic nephropathy: protect glomerular and endothelial function

34

[115]

Grapes, red wine

Phosphorylation of ERK [116]

Proteasomal inhibitor [113] [121]

Abolition of renal and aortic inflammatory response

[122] [123]

Blueberrie s, grapes

Dissociated Keap1Nrf2 complex by phosphorylation of Nrf2 [124]

Diabetic retinopathy: Reduce retinal inflammation

[126]

Diabetic nephropathy: Reduces urinary protein [127] excretion, serum creatinine and blood urea nitrogen

lP

Interacts with the basic amino acids of kelch domain of Keap1 [76]

ro

Pterostilbene

peptide aldehyde

-p

MG132

Diabetic cardiomyopathy: protects diabetes damaged cardiac tissues

of

Inhibits proteasome thereby preventing the degradation of Nrf2 [117]

Diabetic retinopathy: Preserve eNOS phosphorylation [118] in retinal cells [119] Diabetic nephropathy: Alleviate glomerular [120] basement membrane thickening and changes in the proximal and convoluted tubules

re

Resveratrol

Jo

ur na

Activation of AMPK/Akt/GSK3β signaling pathway [125]

35